Scientific Journal Article experiment question

[JustinM88]

So I officially read my first full scientific journal article in preparation for the MCAT and I have a question on the very last experiment that these researchers did. Feel free to correct me on anything and everything that I type here because I'm still new to this whole reading of scientific journal articles thing lol.


Main Idea of the Article
The 3' UTR region of the KLF6 gene (a tumor suppressor gene) is implicated in the downregulation of KLF6 gene expression. Of course, if we downregulate a tumor suppressor gene (aka we weaken the brakes on cell cycle regulation), we can end up with cancer (and in this case, liver cancer).


The Experiment
What the researchers wanted to know:
If the decrease in protein expression (indicated by Luciferase Activity) of the KLF6 gene was due to
a) mRNA degradation
-or-
b) blocking of protein translation.
*(I'm assuming miRNA's were the cause of either of these scenarios?)

Independent Variables:
A control cell (does NOT have KLF6 3' UTR, so I believe this should indicate normal levels of mRNA expression)
A cell w/ a full length 3' UTR (b/c the 3' UTR was shown to decrease protein expression)
A cell w/ UTR segment #2 only (b/c this segment was shown to have the most impact on a decrease in protein expression compared to the other UTR segments)

Dependent Variable:
Luciferase gene expression (% of the control cell) via RT-PCR + qPCR
(I believe RT-qPCR essentially indicates the amount of mRNA present in each cell b/c RT-PCR reverse transcribes RNA back into cDNA & qPCR can quantify cDNA...so overall this indicates how much mRNA there was)

Figure C (the Results)

Quote from the scientific journal article:
"As shown in Figure 4C, the construct containing KLF6 3'UTR segment 2 reduced luciferase mRNA by 50% as compared to control further indicating that KLF6 3'UTR affects the expression of the reporter gene at the post-transcriptional level."

Conclusion:
??? This is where I have a question. Was the answer to the researchers question a) mRNA degradation -or- b) blocking of protein translation ( or c) we still don't really know ) ? I am leaning towards 'a) mRNA degradation' being the cause of the decrease in protein expression since the graph shows that there were less amounts of mRNA when any 3' UTR was present in a cell.
If the answer to the researchers' question was 'b) blocking of protein translation', would each of these bars in the graph be equal to each other since maybe RT-qPCR would count up the same amounts of mRNA in each of these cells since if the miRNA's merely 'blocked translation' this would mean the mRNA was not degraded, and therefore PCR would still quantify the mRNA in each cell as being about the same...?

Source: The Role of the 3' Untranslated Region in the Post-Transcriptional Regulation of KLF6 Gene Expression in Hepatocellular Carcinoma

---

Members don't see this ad.

 

[NextStepTutor_1]

Hi @JustinM88 -

Very cool that you took the plunge into reading scientific articles . To answer your question more specifically, let me summarize what's going on here.

The researchers conducted an exploratory study (the words "we provide the first steps towards better understanding of..." in the abstract point this out) of how KLF6 expression in HCC is regulated. In other words, they did a few separate experiments aimed at getting a general understanding of this issue, instead of testing a specific hypothesis. Molecular biology papers like this often contain multiple sub-experiments, each of which is presented in a figure. So let's work through Fig. 1-4 to see what they did.

Fig. 1 shows findings about how the KLF6 gene is organized.

Fig. 2 shows the half-life of KLF6 mRNA in various cell lines. Based on the abstract and discussion, it seems like the noteworthy point here is that KLF6 mRNA "was highly unstable in liver cancer-derived cell lines as compared to normal hepatocytes". Okay. We don't really know the significance of this point yet, but we can move on.

Fig. 3 tells us about how they used luciferase-encoding vectors containing the KLF6 3'UTR to figure out how specifically the UTR affects things. To understand the point of this, we have to understand what luciferase does, which they don't explain in detail -- they just assume you will understand it, although they give you some clues about the "luciferase reporter gene". Basically, luciferase is a bioluminescent enzyme used to track transcriptional activity in genetic engineering contexts. So what we see in Fig. 3A is that luciferase activity plummeted in the cells with the KLF6 3'UTR. Now they have to ask themselves why this happened, and this is where the researchers pose the question you noted ("We next asked whether KLF6 UTR inhibitory effect on luciferase activity was due to inhibition of mRNA expression or repression of translation."). An important point here is that this question applies to this subexperiment, not to the main goal of the study as a whole. This question is answered in Fig. 3B, which shows that the 3' UTR affected the transcription of the reporter gene.

Now the researchers wondered about which parts of the 3' UTR led to these effects, and information about this question is presented in Fig. 4A-C.

So, in your specific question, it seems like the confusion is coming because you're trying to use Figure 4C to answer a question about the information in Figure 3B.

In general, be sure to keep an eye out for distinguishing the main point of a study and the various subexperiments that the researchers may use to develop their line of reasoning. When you see multiple figures in the results, many of which contain subfigures of their own, that's a clue that this is a study composed of multiple distinct experiments that have both a logical structure of their own that you have to understand and a relationship with the larger chain of reasoning.

Hope this helps & best of luck!!

 

[JustinM88]

Hi @JustinM88 -

Very cool that you took the plunge into reading scientific articles . To answer your question more specifically, let me summarize what's going on here.

The researchers conducted an exploratory study (the words "we provide the first steps towards better understanding of..." in the abstract point this out) of how KLF6 expression in HCC is regulated. In other words, they did a few separate experiments aimed at getting a general understanding of this issue, instead of testing a specific hypothesis. Molecular biology papers like this often contain multiple sub-experiments, each of which is presented in a figure. So let's work through Fig. 1-4 to see what they did.

Fig. 1 shows findings about how the KLF6 gene is organized.

Fig. 2 shows the half-life of KLF6 mRNA in various cell lines. Based on the abstract and discussion, it seems like the noteworthy point here is that KLF6 mRNA "was highly unstable in liver cancer-derived cell lines as compared to normal hepatocytes". Okay. We don't really know the significance of this point yet, but we can move on.

Fig. 3 tells us about how they used luciferase-encoding vectors containing the KLF6 3'UTR to figure out how specifically the UTR affects things. To understand the point of this, we have to understand what luciferase does, which they don't explain in detail -- they just assume you will understand it, although they give you some clues about the "luciferase reporter gene". Basically, luciferase is a bioluminescent enzyme used to track transcriptional activity in genetic engineering contexts. So what we see in Fig. 3A is that luciferase activity plummeted in the cells with the KLF6 3'UTR. Now they have to ask themselves why this happened, and this is where the researchers pose the question you noted ("We next asked whether KLF6 UTR inhibitory effect on luciferase activity was due to inhibition of mRNA expression or repression of translation."). An important point here is that this question applies to this subexperiment, not to the main goal of the study as a whole. This question is answered in Fig. 3B, which shows that the 3' UTR affected the transcription of the reporter gene.

Now the researchers wondered about which parts of the 3' UTR led to these effects, and information about this question is presented in Fig. 4A-C.

So, in your specific question, it seems like the confusion is coming because you're trying to use Figure 4C to answer a question about the information in Figure 3B.

In general, be sure to keep an eye out for distinguishing the main point of a study and the various subexperiments that the researchers may use to develop their line of reasoning. When you see multiple figures in the results, many of which contain subfigures of their own, that's a clue that this is a study composed of multiple distinct experiments that have both a logical structure of their own that you have to understand and a relationship with the larger chain of reasoning.

Hope this helps & best of luck!!

I appreciate that you took the time to look over this!

Now, I have a hard time believing that I was using Figure 4C to answer a question in 3B because the article literally says this:
“To address whether the decrease in the luciferase activity observed is due to mRNA degradation or blocking of protein translation, we quantified luciferase mRNA by RT-qPCR in transfected cells. As shown in Figure 4C, ...”

So, was the decrease in Luciferase activity due to a) mRNA degradation -or- b) blocking of translation... and what part of the graph indicates the answer?? Or, maybe they never actually got the right data to get such a detailed answer to this specific of a question?

 

[NextStepTutor_1]

Hi @JustinM88,

Yes! I see what you're saying. Sorry about the confusion; they also address the very similar-sounding question of "whether KLF6 UTR inhibitory effect on luciferase activity was due to inhibition of mRNA expression or repression of translation" in the discussion of Figure 3, and I think I interpreted your question as referring to that. In any case, it looks like Fig. 4C is very similar to Fig. 3B, with the additional twist that they distinguished between the full UTR and segment/fragment #2. To be honest, I'm a little puzzled at why they're presenting such similar information in both figures.

In any case, the basic idea seems to still be that they observed a low level of the luciferase protein in certain cultures in Fig. 4B and confirmed that this corresponded to a lower level of mRNA, which would support the idea that the findings were due to inhibited mRNA expression, rather than repressed translation. If the problem were at the translation level, you'd expect an unchanged amount of mRNA that just isn't being translated into the luciferase protein for whatever reason.

Hope this clarifies things slightly better!

 

[JustinM88]

Hi @JustinM88,

Yes! I see what you're saying. Sorry about the confusion; they also address the very similar-sounding question of "whether KLF6 UTR inhibitory effect on luciferase activity was due to inhibition of mRNA expression or repression of translation" in the discussion of Figure 3, and I think I interpreted your question as referring to that. In any case, it looks like Fig. 4C is very similar to Fig. 3B, with the additional twist that they distinguished between the full UTR and segment/fragment #2. To be honest, I'm a little puzzled at why they're presenting such similar information in both figures.

In any case, the basic idea seems to still be that they observed a low level of the luciferase protein in certain cultures in Fig. 4B and confirmed that this corresponded to a lower level of mRNA, which would support the idea that the findings were due to inhibited mRNA expression, rather than repressed translation. If the problem were at the translation level, you'd expect an unchanged amount of mRNA that just isn't being translated into the luciferase protein for whatever reason.

Hope this clarifies things slightly better!

Yeah, I saw that other similar info as well. I, too am confused as to why did such similar things haha. I notice you are maybe hesitant to officially declare if the decrease in mRNA was due to 'degradation'...are you equating "inhibited mRNA expression" to "degradation of mRNA"? Or, do you think further experiments need to be done to officially declare it as 'mRNA degradation'?

I do want to say I appreciate you taking the time to look over this with me and that seeing you and Altius Premier Tutor actually discussing these things at this level makes me trust yalls company more than some larger companies that will not be named ^.^

 

[NextStepTutor_1]

Hmm, in general I try to be cautious about concluding that a specific mechanism is supported, but the points made in the discussion section make it sound very likely that mRNA degradation took place.

And thanks! That's great to hear. I can definitely confirm that we at Next Step are real people and we really are invested in our students' success .